The prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine among pregnant women at first antenatal clinic attendance and delivery in the forest-savannah area of Ghana

Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) is used to prevent malaria and associated unfavorable maternal and foetal outcomes in pregnancy in moderate to high malaria transmission areas. Effectiveness of IPTp-SP is, however, threatened by mutations in the Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthase (Pfdhps) genes which confer resistance to pyrimethamine and sulfadoxine, respectively. This study determined the prevalence of molecular markers of SP resistance among pregnant women in a high malaria transmission area in the forest-savannah area of Ghana. Genomic DNA was extracted from 286 P. falciparum-positive dried blood spots obtained from pregnant women aged ≥18 years (255 at first Antenatal Care (ANC) clinic visit and 31 at delivery from 2017 to 2019) using Chelex 100. Mutations in Pfdhfr and Pfdhps genes were detected using molecular inversion probes and next generation sequencing. In the Pfdhfr gene, single nucleotide polymorphisms (SNPs) were detected in 83.1% (157/189), 92.0% (173/188) and 91.0% (171/188) at codons 51, 59, and 108 respectively in samples collected at first ANC visit, while SNPs were detected in 96.6 (28/29), 96.6% (28/29) and 96.8% (30/31) in isolates collected at delivery. The Pfdhfr triple mutant N51I, C59R and S108N (IRN) was carried by 80.5% (128/159) and 96.5% (28/29) of the typed isolates collected at ANC visit and at delivery respectively. In the Pfdhps gene, SNPs were detected in 0.6% (1/174), 76.2% (138/181), 33.2% (60/181), 1.2% (2/174), 0% (0/183), and 16.6% (27/173) at codons 431, 436, 437, 540, 581 and 613 respectively in samples collected at ANC, and 0% (0/25), 72% (18/25), 40% (10/25), 3.6% (1/25), 0% (0/29) and 7.4% (2/27) in samples collected at delivery. Quadruple mutant Pfdhfr N51I, C59R, and S108N + Pfdhps A437G (IRN-GK) was present in 25.8% (33/128) and 34.8% (8/23) of isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forest-savannah area in the middle belt of Ghana.

isolates at ANC and at delivery respectively. Quintuple mutant alleles Pfdhfr N51I, C59R, and S108N + Pfdhps A437G and K540E (IRN-GE) were detected in 0.8% (1/128) and 4.4% (1/23) of samples collected at ANC and at delivery respectively. No mutations were identified at Pfdhfr codons 16 or 164 or Pfdhps 581. There is a high prevalence of Pfdhfr triple mutant P. falciparum infections among pregnant women in the study area. However, prevalence of the combined Pfdhfr/Pfdhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutant was detected, indicating that SP is still likely to be efficacious for IPTp-SP in the forestsavannah area in the middle belt of Ghana.

Background
An estimated 241 million malaria cases were reported globally in 2020, with 228 million cases (95%) occurring in the World Health Organization (WHO) African Region [1]. Malaria during pregnancy remains a major public health problem, with an estimated 11.6 million out of 33.8 million (34%) pregnancies exposed to malaria infection in areas of moderate to high malaria transmission in Africa, resulting in 819,000 children with low birthweight in 2020 [1].
To minimize the potential unfavorable maternal and foetal outcomes of malaria, such as maternal anaemia, low birthweight, preterm delivery and stillbirth associated with malaria during pregnancy in areas of moderate to high transmission of malaria [2, 3], the WHO recommends the use of intermittent preventive treatment using sulfadoxine-pyramethamine (IPTp-SP) starting early in the second trimester, and given at intervals of at least one month apart until as close as possible to delivery [4]. Efficacy of IPTp-SP is influenced by the level of resistance to SP in the Plasmodium falciparum parasite populations [5,6], and the level of resistance is also influenced by SP use in the population [7,8]. Increasing resistance of P. falciparum to SP is attributable to point mutations in the dihydrofolate reductase (Pfdhfr) gene at codons N51I, C59R, S108N and I164L and in the dihydropteroate synthase (Pfdhps) gene at codons S436A, A437G, K540E, A581G and 613T/S, which have been reported to confer resistance to pyrimethamine and sulfadoxine respectively [9][10][11]. Mutations in the Pfdhfr and Pfdhps genes usually occur in a progressive manner [12]. Accumulation of mutations in codons 51, 59 and 108 of the Pfdhfr gene (IRN triple mutation) combined with mutations in the Pfdhps gene at codon 437 are referred to as a quadruple mutation (IRN-GK) which confers partial resistance to SP; the triple Pfdhfr mutation combined with the double Pfdhps mutations A437G and K540E results in a quintuple mutation (IRN-GE) which confers full resistance; the triple Pfdhfr mutation and triple Pfdhps mutations (A437G, K540E and A581G) gives a sextuple mutation (IRN-SGEGA) which confers super resistance [13].
Increasing prevalence of mutations in the Pfdhfr and Pfdhps genes of P. falciparum parasites threatens the use of SP for preventing malaria during pregnancy. WHO recommends that IPTp-SP implementation can be continued in areas of moderate to high malaria transmission when the prevalence of K540E and A581G mutations are <95% and <10%, respectively [14]. Continuous surveillance of SP resistance markers is, therefore, very important to identify any accumulation of mutations in the Pfdhfr and Pfdhps genes of P. falciparum to guide policy on use of SP for IPTp programmes.
In East and Southern Africa, very high levels of quintuple and sextuple mutations have been reported among pregnant women, children and in the general population, almost reaching 100% saturation in some areas and resulting in loss of effectiveness of IPTp-SP [6, [15][16][17]. In West and Central Africa, however, Pfdhfr triple mutation and Pfdhps S436A and A437G mutations are common, but the presence of the K540E and A581G mutations is rare [18][19][20][21][22]. In Ghana, a few studies on SP resistance markers among pregnant women from different locations attending ANC showed IRN, IRN-SGKAA and IRN-SGEAA mutations covering a range from 71% to 80%, 12-41%, and 0-1% [23-25]. There are data for SP resistance markers in children resident in the forest-savannah zone of Ghana [26,27] but there are no published data on SP resistance markers in P. falciparum isolates obtained from pregnant women in this region.
This study aimed at determining current levels of circulating mutations and haplotypes in the Pfdhfr and Pfdhps genes among pregnant women prior to commencement of IPTp-SP and at delivery, in an area of high malaria transmission in the forest-savannah transition zone in the middle belt of Ghana.

Study area, participants and sample collection
The study area has been described previously [28]. Briefly, the study was conducted in four adjoining administrative areas, namely the Kintampo North Municipality, Kintampo South District, Nkoranza North District and Nkoranza South Municipality, all within the forestsavannah transitional ecological zone in the middle belt of Ghana. Anopheles gambiae and An. funestus are the main vectors of malaria transmission in the area. Malaria transmission is high and perennial, with a peak in each year between April and October [27,29].
This study was part of a trial that evaluated the effectiveness of four or more doses of IPTp-SP in the middle belt of Ghana. Dried blood spot (DBS) samples from a cohort of pregnant women were collected at their first antenatal care (ANC) clinic visit (prior to administration of IPTp-SP) [28] and at delivery [30] to determine the prevalence of molecular markers of resistance to SP.
The study procedures have been described previously [28]. Briefly, informed consent was obtained from pregnant women aged 18 years or above who were visiting the ANC for the first time. A structured questionnaire was completed followed by collection of a venous blood sample (2 mL) into EDTA vacutainer tubes prior to commencement of SP administration. At delivery, 0.2 mL of peripheral and placental blood samples were collected into EDTA microtainer tubes. Thick and thin blood smears were prepared from each sample on the same slide, stained with 10% Giemsa stain after fixing the thin film with absolute methanol and examined by two independent, certified malaria microscopists; discrepancies in presence/absence, species and density were resolved by a third microscopist, according to the method of Swysen et al. [31] and WHO [32]. From EDTA anticoagulated blood samples collected from each participant prior to commencement of IPTp-SP and at their time of delivery, three 50 μL blood spots were preserved on 3MM Whatman filter paper (GE Healthcare, Boston, MA, USA) by air-drying overnight and storing individually with silica gel dessicant in a ziplock bag at room temperature until molecular testing was performed. The DBS from all women who tested positive for P. falciparum malaria by microscopy prior to IPTp-SP commencement and/or at delivery were selected for DNA extraction and molecular inversion probe sequencing [33, 34] for markers of Pfdhfr and Pfdhps gene mutations. P. falciparum molecular inversion probe (MIP) design, MIP capture, amplification, sequencing and data processing were performed as previously described [33][34][35]. Sensitivity and accuracy of the MIP captures were determined using serial dilutions of a control mixture of DNA isolated from the laboratory strains of P. falciparum 3D7 (wild-type), HB3, 7G8, and DD2 (mutants) which were mixed at relative frequencies of 67%, 14%, 13%, and 6%, respectively.
Drug resistance markers (amino acid mutations determined from the underlying DNA sequence) examined for this study were Pfdhfr A16V, N51I, C59R, S108N/T, I164L for pyrimethamine, and Pfdhps A431V, S436A, A437G, K540E, A581G and A613S for sulfadoxine. All genotypes with >1 unique molecular identifiers (UMI) coverage were selected for downstream analysis. Results were classified as wild type or mutant. Samples containing mixed infections (i.e. both wild type and mutant) were considered as mutants. Samples with mutations 51I+59R +108N/T (IRN) of the Pfdhfr protein and 437G+540E (GE) of the Pfdhps protein were classified as Pfdhfr triple mutants and Pfdhps double mutants respectively. Overall classification combined the number of mutations in both Pfdhfr and Pfdhps genes. Triple Pfdhfr mutants + 437G, Pfdhfr triple mutant + Pfdhps double mutant were classified as quadruple (IRN-GK) and quintuple (IRN-GE) mutants respectively.

Statistical analysis
Data from the sequence analysis were stored in Microsoft Excel. Statistical analysis was performed using Stata 14 (StataCorp, College Station, TX, USA). Data were presented for samples collected at ANC clinic prior to commencement of IPTp-SP or at delivery. Prevalence of SP resistance mutations was defined as the percentage of pregnant women who carried at least one resistant parasite clone [36]. The percentage of mutations in the Pfdhfr and Pfdhps genes at each time point were calculated as the number of samples with mutation at a specific codon divided by the number of samples successfully genotyped and multiplied by 100, whiles percentage of haplotypes were calculated as the haplotype divided by the number of samples with complete genotype results for the haplotype, multiplied by 100. Differences in carriage of SNPs between samples collected prior to commencement of IPTp-SP and at delivery was assessed using the Chi-square test. A p-value of <0.05 was considered significant.

Ethical considerations
Ethical approval for the study was obtained from the ethics committees of the Kintampo Health Research Centre (KHRCIEC/2017-9) and the London School of Hygiene and Tropical Medicine (LSHTM Ethics Ref: 12338). Informed consent was obtained from each participant prior to enrolment into the study.

Characteristics of study participants
Molecular characterization of resistance markers was undertaken successfully in at least one codon for 255 (80%) of 317 DBS samples from women with malaria parasitaemia at first ANC clinic visit and for 31 (94%) of 33 women with a malaria infection at delivery. Samples for which characterization of resistance markers was unsuccessful was due to inadequate amounts of parasite DNA at time of analysis. There was, however, no difference in characteristics of study participants at enrollment whose resistance markers were successfully characterized and those that were not characterized (S1 Table). About a half of the pregnant women at enrolment or delivery were aged <24 years old. Malaria parasite density range (Geometric Mean Parasite Density) at enrolment and delivery were 20-46500 (522) and 82-13246 (547) parasites per microlitre of blood, respectively. Mean number of SP doses taken at time of delivery was 2.9 (SE 0.21) ( Table 1).

PLOS ONE
Prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine in forest-savannah area of Ghana
Complete Pfdhps codons 436/437/540/581/613 haplotypes were available for 127 and 22 samples collected at ANC and at delivery respectively (Table 3). Ten (10) and 5 distinct dhps haplotypes were obtained for samples collected at ANC and at delivery respectively. Wildtype haplotype of Pfdhps, SAKAA, was present in 2.4% (3/127) of P. falciparum isolates at ANC whiles none was detected in isolates at delivery. The double mutant SGEAA responsible for sulfadoxine resistance was detected in 0.8% (1/127) of samples collected at first ANC, and 4.5% (1/22) among those collected at delivery (Table 3).

PLOS ONE
Prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine in forest-savannah area of Ghana Of the 31 samples collected at delivery, 7 had a positive malaria microscopy result at both first ANC clinic visit and at delivery. The complete genotype for the Pfdhfr codons 51, 59 and 108 was obtained for 4 sample pairs (prior to IPTp-SP and at delivery), with all 4 pairs having the triple mutant IRN (100% correlation between the two timepoints) whiles complete genotype for the Pfdhps codons 437 and 540 was obtained for 3 sample pairs as AK/GK, AK/AK and GK/GK.

Prevalence of combined P. falciparum dhfr and dhps haplotypes
A total of 128 and 23 samples had a complete set of genotyped results for the Pfdhfr codons 51/ 59/108 and Pfdhps codons 437/540 haplotypes at ANC and at delivery respectively. Eleven (11) distinct Pfdhfr haplotypes were observed in samples collected at ANC whiles 4 were observed for samples collected at delivery. The IRN-AK haplotype constituted the majority in samples collected at ANC (53.1%, 68/128) and at delivery (56.5%, 13/23), followed by the quadruple mutant IRN-GK which was 25.8% (33/128) at ANC and 34.7% (8/23) at delivery. The quintuple IRN-GE mutation was detected in 0.8% (1/128) and 4.4% (1/23) in samples collected at ANC and at delivery respectively. The wildtype haplotype NCS-AK was present in 5.5% (7/ 128) at ANC but not was detected at delivery (Fig 1).

PLOS ONE
Prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine in forest-savannah area of Ghana Complete data for the combined Pfdhfr 51/59/108 and Pfdhps 436/437/540/581/613 was available for 114 and 22 samples collected at ANC and at delivery respectively (Table 4). A total of 21 and 6 distinct haplotypes were obtained from the combined Pfdhfr and Pfdhps haplotypes. The wild type haplotype (NCS-SAKAA) were carried by 1.7% (2/114) of isolates at

PLOS ONE
Prevalence of molecular markers of resistance to sulfadoxine-pyrimethamine in forest-savannah area of Ghana first ANC visit and none at delivery. Carriage of the quadruple mutant IRN-SGKAA was 10.5% (12/114) at first ANC visit and 18.2% (4/22) in the isolates collected at delivery. Triple mutant IRN combined with S436A mutation alone represented the highest proportion of mutants (>40%) at both timepoints (Table 3). Quintuple mutations associated with codon 540 (IRN-SGEAA) were detected in 0.9% (1/112) and 4.6% (1/22) of samples at ANC and at delivery.

Discussion
Surveillance for P. falciparum dhfr and dhps resistance markers to sulfadoxine-pyrimethamine among pregnant women is important in evaluating the potential effectiveness of IPTp-SP and in informing policy on malaria control during pregnancy. The recommended approaches for monitoring drug resistance are (i) in vivo drug efficacy estimates based on parasite clearance, (ii) in vitro/ex vivo drug efficacy assays, and (iii) genotyping of molecular markers. Although determination of molecular markers of resistance is more expensive, time-consuming and laborious compared to in vitro and in vivo assays for determination of drug resistance, it serves as an excellent complement to the in vitro and in vivo approaches [37,38]. This study evaluated carriage of markers of resistance to sulfadoxine and pyrimethamine by describing SNPs in codons 51, 59, 108 and 164 of the Pfdhfr gene and in codons 431, 436, 437, 540, 581 and 613 of the Pfdhps gene, as well as haplotypes in the Pfdhfr, Pfdhps and combined Pfdhfr/Pfdhps genes among pregnant women prior to commencement of IPTp-SP and at delivery in an area of high malaria transmission in the forest-savannah zone in the middle belt of Ghana to serve as a baseline for monitoring molecular markers of resistance to SP. It has been proposed that resistance frequencies, as well as prevalence measures, should be evaluated for policy decisions. Whiles prevalence of resistance mutations represent the proportion of infected individuals carrying at least one resistant parasite, frequency of resistance represent the proportion of parasite clones which is carrying a resistant marker [36]. Prevalence of resistance was selected for the analysis of this study as it is a more important way to examine the data for clinical drug effectiveness.
There is no published data on the prevalence of resistance markers to SP among pregnant in the studied area. However, compared to results from samples previously collected from children in the study area, the current study showed a higher prevalence in Pfdhfr SNPs at codons 51, 59 and 108 than in Only a small fraction of isolates harboured P. falciparum parasites with the A437G mutation in the Pfdhps gene in combination with Pfdhfr triple mutation, which has been reported to be associated with SP treatment failure. This 26% is much lower compared to other studies in  [18,40,42]. This is an indication that IPTp-SP may still be effective in the study area.
Although not statistically significant, the increase in prevalence of point mutations at codons 51, 59, 108 of the dhfr gene and at codons 437 and 540 of the dhps gene in samples collected at delivery compared to those collected prior to commencement of IPTp-SP in this study is consistent with other studies [25,48]. This increase could be suggestive of increased selection of SP-resistant P. falciparum parasites following SP supplementation during pregnancy [25,48], or new infections with any of the resistant genotype circulating in the studied area. The average number of SP doses received by the thirty-one women during pregnancy in the study who had malaria parasitaemia at delivery was nearly three doses (slightly below the optimal dose of �3 doses). Non-compliance with optimal SP dosing is an important contributor to the emergence of drug-resistant P. falciparum strains [49]. Increased use of SP has been reported to be associated with clearance of the sensitive P. falciparum strains, selection and increased circulation of the resistant phenotype which can be transferred to future generations of the parasite [50,51].
Detection of the I431V mutation in this study, albeit at a low prevalence, supports a previous finding of the mutation in samples of Ghanaian immigrants in China . These levels of mutations may impact on the use of SP for IPTp. Continuous testing will be necessary to monitor possible distribution and levels of these mutations.
A major strength of this study is that it has described the level of Pfdhfr and Pfdhps molecular markers of resistance to SP among pregnant women in this area of high malaria transmission which hitherto had not been performed. This study thus provides baseline data for monitoring SP resistance markers in the study area. The study also used the MIPs followed by Next Generation Sequencing which is a highly sensitive and high throughput method for the DNA amplification stage. A limitation, however, is that the number of P. falciparum infections at delivery was small and could have resulted in low statistical power to detect an increased prevalence of SP resistance markers following IPTp-SP administration. This reduction in infection numbers itself (i.e. about 20% prevalence of parasitaemia prior to commencement of IPTp-SP [28] against about 3% prevalence at delivery [30] in the study area) suggests the effectiveness of IPTp-SP in clearing P. falciparum infections during pregnancy. Also, DNA could not be amplified in a substantial number of DBS, possibly due to DNA degradation. However, the baseline characteristics did not differ between the women's whose samples were successfully sequenced and those that were not.

Conclusions
There is a high prevalence of Pfdhfr triple mutants in the forest-savannah zone in the middle belt of Ghana, but this has not reached saturation levels in pregnant women at their first ANC visit. However, prevalence of the combined dhfr/dhps quadruple and quintuple mutants IRN-GK and IRN-GE respectively prior to commencement of IPTp-SP were low, and no Pfdhps A581G mutations were identified in this study. The findings indicate that SP is still efficacious for use as IPTp in the forest-savannah zone, a high malaria transmission area in the middle belt of Ghana.
Supporting information S1 Table. Socio-demographic characteristics of pregnant women with malaria parasitaemia at first antenatal care clinic visit whose dried blood spot samples were used in the study and of those women from whom no data for analysis were obtained.